The adenoviruses cause enteric or respiratory infection in humans as well as in domestic and laboratory animals.
The bovine adenoviruses (BAVs) comprise at least nine serotypes divided into two subgroups. These subgroups have been characterized based on enzyme-linked immunoassays (ELISA), serologic studies with immunofluorescence assays, virus-neutralization tests, immunoelectron microscopy, by their host specificity and clinical syndromes. Subgroup 1 viruses include BAV 1, 2, 3 and 9 and grow relatively well in established bovine cells compared to subgroup 2 which includes BAV 4,5,6,7 and 8.
BAV3 was first isolated in 1965 and is the best characterized of the BAV genotypes, containing a genome of approximately 35 kb (Kurokawa et al (1978) J. Virol. 28:212-218). BAV3, a representative of subgroup 1 of BAVs (Bartha (1969) Acta Vet. Acad. Sci. Hung. 19:319-321), is a common pathogen of cattle usually resulting in subclinical infection (Darbyshire et al. (1965). J. Comp. Pathol. 75:327-330), though occasionally associated with a more serious respiratory tract infection (Darbyshire et al., 1966 Res. Vet Sci 7:81-93; Mattson et al., 1988 J. Vet Res 49:67-69). Like other adenoviruses, BAV3 is a non-enveloped icosahedral particle of 75 nm in diameter (Niiyama et al. (1975) J. Virol. 16:621-633) containing a linear double-stranded DNA molecule. BAV3 can produce tumors when injected into hamsters (Darbyshire, 1966 Nature 211:102) and viral DNA can efficiently effect morphological transformation of mouse, hamster or rat cells in culture (Tsukamoto and Sugino, 1972 J. Virol. 9:465-473; Motoi et al., 1972 Gann 63:415-418; M. Hitt, personal communication). Cross hybridization was observed between BAV3 and human adenovirus type 2 (HAd2) (Hu et al., 1984 J. Virol. 49:604-608) in most regions of the genome including some regions near but not at the left end of the genome.
In the human adenovirus (HAd) genome there are two important regions: E1 and E3 in which foreign genes can be inserted to generate recombinant adenoviruses (Berkner and Sharp (1984) Nuc. Acid Res., 12:1925-1941 and Haj-Ahmad and Graham (1986) J. Virol., 57:267-274). E1 proteins are essential for virus replication in tissue culture, however, conditional-helper adenovirus recombinants containing foreign DNA in the E1 region, can be generated in a cell line which constitutively expresses E1 (Graham et al., (1977) J. Gen. Virol., 36:59-72). In contrast, E3 gene products of HAd 2 and HAd 5 are not required for in vitro or in vivo infectious virion production, but have an important role in host immune responses to virus infection (Andersson et al (1985) Cell 43:215-222; Burgert et al (1987) EMBO J. 6:2019-2026; Carlin et al (1989) Cell 57:135-144; Ginsberg et al (1989) PNAS, USA 86:3823-3827; Gooding et al (1988) Cell 53:341-346; Tollefson et al (1991) J. Virol. 65:3095-3105; Wold and Gooding (1989) Mol. Biol. Med. 6:433-452 and Wold and Gooding (1991) Virology 184:1-8). The E3-19 kiloDalton (kDa) glycoprotein (gp19) of human adenovirus type 2 (HAd2) binds to the heavy chain of a number of class 1 major histocompatibility complex (MHC) antigens in the endoplasmic reticulum thus inhibiting their transport to the plasma membrane (Andersson et al. (1985) Cell 43:215-222; Burgert and Kvist, (1985) Cell 41:987-997; Burgert and Kvist, (1987) EMBO J. 6:2019-2026). The E3-14.7 kDa protein of HAd2 or HAd5 prevents lysis of virus-infected mouse cells by tumor necrosis factor (TNF) (Gooding et al. (1988) Cell 53:341-346). In addition, the E3-10.4 kDa and E3-14.5 kDa proteins form a complex to induce endosomal-mediated internalization and degradation of the epidermal growth factor receptor (EGF-R) in virus-infected cells (Carlin et al. Cell 57:135-144; Tollefson et al. (1991) J. Virol. 65:3095-3105). The helper-independent recombinant adenoviruses having foreign genes in the E3 region replicate and express very well in every permissive cell line (Chanda et al (1990) Virology 175:535-547; Dewar et al (1989) J. Virol. 63:129-136; Johnson et al (1988) Virology 164:1-14; Lubeck et al (1989) PNAS, USA 86:6763-6767; McDermott et al (1989) Virology 169:244-247; Mittal et al (1993) Virus Res. 28:67-90; Morin et al (1987) PNAS, USA 84:4626-4630; Prevec et al (1990) J. Inf. Dis. 161:27-30; Prevec et al (1989) J. Gen. Virol. 70:429-434; Schneider et al (1989) J. Gen. Virol. 70:417-427 and Yuasa et al (1991) J. Gen. Virol. 72:1927-1934). Based on the above studies and the suggestion that adenoviruses can package approximately 105% of the wild-type (wt) adenovirus genome (Bett et al (1993) J. Virol. 67:5911-5921 and Ghosh-Choudhury et al (1987) EMBO. J. 6:1733-1739), an insertion of up to 1.8 kb foreign DNA can be packaged into adenovirus particles for use as an expression vector for foreign proteins without any compensating deletion.
The E1A gene products of the group C human adenoviruses have been very extensively studied and shown to mediate transactivation of both viral and cellular genes (Berk et al., 1979 Cell 17:935-944; Jones and Shenk, 1979 Cell 16:683-689; Nevins, 1981 Cell 26:213-220; Nevins, 1982 Cell 29:913-919; reviewed in Berk, 1986 Ann. Res. Genet 20:45-79), to effect transformation of cells in culture (reviewed in Graham, F. L. (1984) "Transformation by and oncogenicity of human adenoviruses. In: The Adenoviruses." H. S. Ginsberg, Editor. Plenum Press, New York; Branton et al., 1985 Biochim. Biophys. Acta 780:67-94) and induce cell DNA synthesis and mitosis (Zerler et al., 1987 Mol. Cell Biol. 7:821-929; Bellet et al., 1989 J. Virol. 63:303-310; Howe et al., 1990 PNAS, USA 87:5883-5887; Howe and Bayley, 1992 Virology 186:15-24). The E1A transcription unit comprises two coding sequences separated by an intron region which is deleted from all processed E1A transcripts. In the two largest mRNA species produced from the E1A transcription unit, the first coding region is further subdivided into exon 1, a sequence found in both the 12s and 13s mRNA species, and the unique region, which is found only in the 13s mRNA species. By comparisons between E1A proteins of human and simian adenoviruses three regions of somewhat conserved protein sequence (CR) have been defined (Kimelman et al., 1985 J. Virol. 53:399-409). CR1 and CR2 are encoded in exon 1, while CR3 is encoded in the unique sequence and a small portion of exon 2. Binding sites for a number of cellular proteins including the retinoblastoma protein Rb, cyclin A and an associated protein kinase p33.sup.cdk2, and other, as yet unassigned, proteins have been defined in exon 1-encoded regions of E1A proteins (Yee and Branton, 1985 Virology 147:142-153; Harlow et al., 1986 Mol. Cell Biol. 6:1579-1589; Barbeau et al., 1992 Biochem. Cell Biol. 70:1123-1134). Interaction of E1A with these cellular proteins has been implicated as the mechanism through which E1A participates in immortalization and oncogenic transformation (Egan et al, 1989 Oncogene 4:383-388; Whyte et al., 1988 Nature 334:124-129; Whyte et al, 1988 J. Virol. 62:257-265). While E1A alone may transform or immortalize cells in culture, the coexpression of both E1A and either the E1B-19 k protein or the E1B-55 k protein separately or together is usually required for high frequency transformation of rodent cells in culture (reviewed in Graham, 1984 supra; Branton et al., 1985 supra; McLorie et al., 1991 J. Gen Virol. 72:1467-1471).
Transactivation of other viral early genes in permissive infection of human cells is principally mediated by the amino acid sequence encoded in the CR3 region of E1A (Lillie et al., 1986 Cell 46:1043-1051). Conserved cysteine residues in a CysX.sub.2 CysX.sub.13 CysX.sub.2 Cys sequence motif (SEQ ID NO: 30) in the unique region are associated with metal ion binding activity (Berg, 1986 supra) and are essential for transactivation activity (Jelsma et al., 1988 Virology 163:494-502; Culp et al., 1988 PNAS, USA 85:6450-6454). As well, the amino acids in CR3 which are immediately amino (N)-terminal to the metal binding domain have been shown to be important in transcription activation, while those immediately carboxy (C)-terminal to the metal binding domain are important in forming associations with the promoter region (Lillie and Green, 1989 Nature 338:39-44; see FIG. 3).
The application of genetic engineering has resulted in several attempts to prepare adenovirus expression systems for obtaining vaccines. Examples of such research include the disclosures in U.S. Pat. No. 4,510,245 on an adenovirus major late promoter for expression in a yeast host; U.S. Pat. No. 4,920,209 on a live recombinant adenovirus type 7 with a gene coding for hepatitis-B surface antigen located at a deleted early region 3; European Patent 389 286 on a non-defective human adenovirus 5 recombinant expression system in human cells for HCMV major envelope glycoprotein; WO 91/11525 on live non-pathogenic immunogenic viable canine adenovirus in a cell expressing E1A proteins; and French Patent 2 642 767 on vectors containing a leader and/or promoter from the E3 region of adenovirus 2.
It is assumed that an indigenous adenovirus vector would be better suited for use as a live recombinant virus vaccine in non-human animal species, as compared to an adenovirus of human origin. This requires that regions suitable for insertion of heterologous sequences be identified in the indigenous adenoviral vector, and that compositions and methods for insertion of heterologous sequence, isolation of recombinants and propagation of recombinants be devised. Regions suitable for insertion could include non-essential regions of a viral genome or essential regions, if an appropriate helper function is provided. For example, if, by analogy to HAds, the E3 regions in other adenoviruses are not essential for virus replication in cultured cells, adenovirus recombinants containing foreign gene inserts in the E3 region could be generated.
The selection of a suitable virus to act as a vector for foreign gene expression, the identification of suitable regions as sites for gene insertion, and the construction, isolation and propagation of recombinant virus pose significant challenges to the development of recombinant viral vaccine vectors. In particular, preferred insertion sites will be non-essential for the viable replication of the virus and its effective operation in tissue culture and also in vivo. Moreover, the insertion sites must be capable of accepting new genetic material, whilst ensuring that the virus continues to replicate. An essential region of a virus genome can also be utilized for foreign gene insertion if the recombinant virus is grown in a cell line which complements the function of that particular essential region in trans.
An efficient method for determining suitable insertion sites in a viral genome is to obtain the complete nucleotide sequence of that genome. This allows the various coding regions to be defined, facilitating their possible use as insertion sites. Definition of nonessential noncoding regions would also be revealed by sequence analysis, and these could also be used as potential insertion sites. The nucleotide sequence of certain regions of the BAV-3 genome has been determined. The sequence of the extreme left end of the genome, including the inverted terminal repeat (ITR), packaging signals, E1 and pIX, has been determined by several groups: nucleotides 1-195 (ITR) by Shinagawa et al., 1987, Gene 55:85-93; nucleotides 1-4060 (ITR, packaging signals, E1 and pIX) by Zheng et al., 1994, Virus Research 31:163-186; nucleotides 1-4091 (ITR, packaging signals, E1 and pIX) by Elgadi et al., 1993, Intervirology 36:113-120. (Nucleotide 1 designates the left-most nucleotide of the linear, 34.4 kb BAV-3 genome.) Additional sequences of the BAV-3 genome that have been determined include: nucleotides 5,235-5,891 (major late promoter, Song et al., 1996, Virology 220:390-401); nucleotides 17,736-20,584 (hexon gene, Hu et al., 1984, J. Virology 49:604-608); nucleotides 20,408-21,197 (proteinase gene, Cai et al., 1990, Nucleic Acids Res. 18:5568; and nucleotides 26,034-31,132 (E3 region, pVIII and fibre genes, Mittal et al., 1992, J. Gen. Virol. 73:3295-3300).
One of the many uses to which recombinant viruses and viral genomes could be applied, if they were available, is in the development of recombinant subunit vaccines. Vaccination has proven to be the most effective means for controlling respiratory and enteric viral diseases, especially when live attenuated viral vaccines have been employed. These vaccines, when administered orally or intranasally, induce strong mucosal immunity, which is required to block the initial infection and to reduce the development of disease caused by these viruses. This approach has been extended by using genetically engineered virus genomes (virulence gene-deleted) as vectors to deliver and express genes of other pathogens in vivo. Ertl et al. (1996) J. Immunol 156:3579-3582.
A recombinant viral vector system based on human adenoviruses (HAVs) has recently been developed. Graham et al. (1992) in "Vaccines: New approaches to immunological problems" (R. W. Ellis ed.), Butterworth-Heineman, Stoneham, pp. 363-390. Both replication-defective and replication-competent HAV vectors have been engineered to express various foreign antigens. For review see Grunhaus et al. (1992) Seminar in Virol. 3:237-252; Imler (1995) Vaccine 13:1143-1151. In addition to providing stable foreign gene expression, engineered adenoviruses have been shown to induce humoral, cellular and mucosal immune responses. Buge et al. (1997) J. Virol. 71:8531-8541.
The use of human adenoviruses as vectors for gene therapy has been hampered because of the presence, in the host, of preexisting neutralizing antibodies against HAVs, which may interfere with entry and replication of recombinant virus, and because of the possibility of recombination and/or complementation between recombinant virus and preexisting wild-type HAV in the host. Therefore, animal adenoviruses other than HAV, which are highly species-specific, are being considered as vectors for gene therapy and recombinant vaccines.
Molecular characterization of bovine adenovirus-3 (BAV3) would aid in the development of bovine adenoviruses as live viral vectors for vaccines and gene therapy, in humans and other mammalian species. Recently, the complete DNA sequence and transcriptional map of the BAV3 genome has been reported. This sequence has been disclosed in the parent U.S. patent application Ser. No. 08/880,234, filed Jun. 23, 1997, and in several publications. Baxi et al. (1998) Virus Genes 16:1-4; Lee et al. (1998). Virus Genes 17:99-100; and Reddy et al. (1998) J. Virol. 72:1394-1402.